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REVIEW
Future of RNAi-based therapies for human papillomavirus-associated cervical cancer Jiezhong Chen, Aaron Irving, Nigel McMillan & Wenyi Gu† †Author
for correspondence University of Queensland, UQ Diamantina Institute, R-Wing, Princess Alexandra Hospital, Ipswich Rd, Brisbane QLD 4102, Australia Tel.: +61 732 405 387; Fax: +61 732 405 946;
[email protected]
Over 99% of cervical cancers are associated with infection of high-risk type human papillomaviruses (HPV). These viruses infect epithelial cells lining the cervix and express the early viral genes E6 and E7, which are oncogenes and are primarily responsible for the transformation of the epithelial cells. The continuous expression of those genes is essential for maintenance of the cancer cell phenotype and viability. These viral genes can be silenced using oligonucleotide-based techniques, for example RNAi, antisense RNA and ribozymes. In spite of promising results in vitro and in vivo, in mice, these methods have thus far proved unsuccessful in humans, owing to the lack of an effective delivery system amongst other limitations. In this review we will discuss potential gene-silencing strategies in cervical cancer that would target both viral genes such as E6 and E7, and cellular genes that become deregulated such as E2F, p53, Akt, mTor, NF-κB or Bcl-2. By investigating these approaches we may generate an effective treatment for HPV-induced cervical cancer using gene silencing.
HPV & the carcinogenesis of cervical cancer
Keywords: Akt, Bcl-2, cervical carcinoma, human papillomavirus E6/E7, mTor, NF-κB, p53, RNA interference part of
Human papillomavirus (HPV) is a nonenveloped, dsDNA virus; there are more than 100 types of HPV that infect epithelial cells and cause proliferative lesions. High-risk HPV types are associated with the development of tumors in humans that account for 5.2% of the entire global cancer burden [1]. These cancers include those of the cervix, penis, vulva/vagina, anus, mouth and oropharynx and resulted in 561,100 new cases in 2002 [2]. Cervical cancer is one of the most prevalent cancers caused by HPV infection and is the second most common cancer in women worldwide [3]. Following entry into cervical epithelial cells, the persistent infection of HPV and sometimes the integration of its DNA into the genome of the host cell (occurs in 50% cases of HPV-16 and 94% of HPV-18 infections [4]) are very important in the carcinogenesis of cervical cancer. The continuous expression of the oncogenes E6 and E7 from the virus, or host cells, is mainly responsible for the transformation of epithelial cells and is also crucial for maintaining the phenotype and survival of the cancer cell [5–7]. HPV E6 and E7 oncoproteins target cellular proteins, some of which have been described previously [8,9]. In brief, HPV E6 binds to the tumor suppressor protein p53 via E6 associated proteins (E6 AP) and induces the degradation of this protein through the ubiquitin–proteasome pathway. p53 is a critical protein for protection against propagation of
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DNA damage, by mediating apoptosis and cell cycle arrest responses to DNA damage. HPV E7 binds to the retinoblastoma (Rb) gene product leading to its degradation via ubiquitin–proteasome pathway. Unphosphorylated Rb protein is an important cell cycle regulatory protein which is found in complex with the E2F transcription factors during the G1 phase of the cell cycle. Upon phosphorylation or binding with oncogene products such as E7, the E2F complex is released, which leads to the activation of the transcription factor E2F and continuous cell proliferation. E6 and E7 are therefore critical for the ongoing proliferation of HPV-infected cells. Indeed, HeLa cells, a HPV 18-positive cell line that has HPV genes (including E6 and E7) integrated into their genome, have been in continuous culture for nearly 50 years and are still sensitive to the loss of E6 and E7 expression. Thus, one strategy for the treatment of HPV-induced cervical cancer might be to inhibit oncogenes E6 and E7 or their downstream pathways. Summary of oligonucleotide-based therapies for cervical cancer
Oligonucleotide-based therapy (OBT) may be applied to block the action of E6 and E7 and could therefore, promote cervical cancer cell death [5]. OBT is a technology in which oligonucleotides complementary to target mRNAs or DNA, inhibit or block the expression of the target gene at the transcriptional, or post-transcriptional, level. The aim of OBT is to suppress E6 Future Virol. (2007) 2(6), 587–595
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and E7 gene expression and therefore promote the cervical cancer cell to undergo p53-dependent apoptosis or to enter senescence. The value of OBT compared with current more traditonal treatments, for example, surgery, chemotherapy and radiotherapy, is its specificity at a molecular level. Indeed, when HPV E6 and E7 mRNA is the target of OBT, targeting at one gene can lead to both E6 and E7 genes being silenced simultaneously, achievable because of the bicistronic nature of their expression [10–12]. ShRNA targeting HPV-18 E6 caused growth inhibition of HeLa cells (HPV-18+) but did not affect SiHa cells (HPV-16+) [11]. In the case of antisense RNA, targeting both E6 and E7 [13] or just E7 [14] always resulted in cancer cell growth inhibition or tumor regression. There are three main types of OBTs for HPV infection that have been investigated to date. These are: antisense oligonucleotides, ribozymes and RNA interference (RNAi). The concept of using oligonucleotide-based gene therapy to treat cervical cancer arose in the early 1990s with antisense RNA; ribozymes were used later. Compared with these two methods, RNAi is a rather new technique and has received intensive interest in recent years as a potential therapy for infectious diseases, genetic disorders and cancer. The major similarities and differences among the three types of OBT are summarized in Figure 1. Antisense oligonucleotides are short oligonucleotides (either RNA or DNA) [15–17] thought to work by physically blocking mRNA-ribosome interactions (Figure 1). By contrast, ribozymes function catalytically, consisting of a small RNA molecule with a catalytic core and a targeting domain. The targeting domain hybridizes to its cognate mRNA sequence via Watson–Crick base pairing; the catalytic core then cleaves the mRNA (Figure 1). Initial in vitro results showed that hammerhead ribozymes directed against various HPV18 targets could reduce the level of target RNA to 21% of the control level, whilst leaving the remaining RNA population untouched. Cancer cell growth and foci formation in soft agar decreased, while serum dependency increased, following treatment with hammerhead ribozymes [18,19]. However, hammerhead ribozymes lacked reliability, which led to the development of hairpin ribozymes that exhibited improved efficacy [20]. Both the antisense oligonucleotides and the ribozymes were uneffective in vivo because of their toxic side effects [14] and immunogenic features when delivered by adenovirus [21]. 588
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RNAi has proved to be a powerful tool specifically to silence gene expression in the laboratory and may be useful in the future as a treatment for genetic disorders, viral disease and cancer [22–26]. RNAi is mediated by a cellular protein complex known as RNA-induced silencing complex (RISC) that can bind any mRNA with complementary sequence to siRNAs already present in the cell or added externally, resulting in degradation of the mRNA or blocking its translation (Figure 1). Alternatively, RNAi can block gene expression at the transcriptional level by forming heterochromatin or by promoting DNA methylation [27]. Previous studies have demonstrated that siRNAs or shRNAs delivered by plasmid DNA or by lentivirus vector can suppress HPV-16 or -18 E6 and E7 expression in various cervical cancer cell lines [10,11,28–32]. The studies all show that RNAi can silence E6 and E7 gene expression and promotes cervical cancer cells to undergo apoptosis or senescence. Several in vivo studies have also demonstrated that RNAi treatment can inhibit tumor growth in mice [11,31]. However, as was found to be the case with antisense oligonucleotides in the 1990s, RNAi has brought us no closer to treating cervical cancer in patients. In order to bring RNAi into the clinic to treat cervical cancer and other diseases, we will need to solve problems associated with delivery and patient safety and to minimize any potential side effects; these factors are discussed in detail below. Limitations of OBT in the development of treatments for cervical cancer
Delivering OBT to cancer cells in vivo is difficult, but nanotechnology may provide an answer. Nanoparticles are being explored as a delivery system for cancer and some other diseases [33–35] and are currently in Phase I clinical trial for cancer treatment [101]. Other delivery systems, most notably liposomes, are also giving promising results in delivering oligonucleotides for the treatment of cancer and other diseases [36–39]. However, there have been no reports of the use of liposomal-mediated RNAi delivery against HPV E6 and E7. Even if an effective OBT delivery system is developed, it will still be necessary to confirm that the OBT is as effective in vivo as it was in vitro in downregulating the E6 and E7 genes, and that it produces few severe side effects. One factor that will influence the efficacy of RNAi will be its ability to persist in the body, as a high and lasting OBT dose may be needed to kill all the metastatic cervical cancer cells. Compared future science group
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Figure 1. Mode of action of oligonucleotide-based therapy. shRNA vector
shRNA
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shRNA is introduced into cells with a vector. It is transcribed by the cellular machinery and folded into a hairpin structure that is processed by a nuclease known as DICER, producing siRNA. The antisense strands of the siRNA bind to RISC. The RISC–siRNA complex binds a cellular target mRNA that is complementary to the siRNA. This leads to the cleavage of the target mRNA; the protein it codes for is not translated. Antisense oligodeoxynuleotides can directly bind to the complementary mRNA region, blocking ribosome movement along the mRNA and thus stopping translation. Hammerhead ribozymes consist of two branches that can locate complementary mRNA sequences. The hammerhead ribozyme then catalyzes the cleavage of mRNA, preventing translation. ODN: OligodeoxynucleotidE; RISC: RNA-induced silencing complex.
with antisense oligonucleotides and ribozymes, RNAi agents will probably be more stable because of their double-strand structure. Given the stability and proven effectiveness of RNAi in vitro, it does, in our view, represent the most promising potential approach to treat cervical cancer. However, RNAi is far from perfect. One issue that may need to be considered is the toxicity of shRNA. shRNAs have been demonstrated to be toxic in vivo; a property that appears to be related to saturation of the endogenous microRNA pathway [40]. In addition, a recent review by Barik described problems associated with shRNA overexpression suggesting interference with the transport from nuclei to the cytoplasm because of the competing use of exportin 5 with miRNA [41]. future science group
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Another important issue to consider is the propensity of cervical cancer cells to develop resistance to RNAi. It is known that HIV strains resistant to RNAi emerge after siRNA treatment [42]. While HPV is less prone to mutation than HIV, Tang et al. recently reported resistance to E7-specific siRNA treatment in cervical cancer cells (Caski and SiHa) [43]. Resistance appeared to be due to a previously unknown 50 kDa cytoplasmic protein that unwound the antisense strand of E7 siRNA from the RISC complex, thereby blocking RNAi. Such resistance in cervical cancer cells would be an obstacle to the development of RNAi-based treatments and might possibly also apply to other forms of OBT. We found that HeLa cells became shRNA resistant after 2 weeks of treatment with 589
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shRNA specific to E7, delivered with a lentiviral vector [Gu et al., Unpublished Data]. Hence, we suggest that resistance may be one of the most important potential obstacles to OBT for the treatment of cervical cancer. It might perhaps be overcome by simultaneously targeting several different viral genes besides E6 and E7, thus reducing the chance of developing resistance to a specific siRNA. Inhibition of major signal pathways in the carcinogenesis of cervical cancer
In HPV-induced cervical cancer, E6 inhibits the p53 pathway while E7 inhibits the pRb pathway (Figure 2). In addition, protein kinase B (Akt) and its downstream proteins, mammalian target of rapamycin (mTor), nuclear factor (NF)-κB, and B-cell leukemia (Bcl)2 are increased, in common with many other cancers. Defects in several distinct signaling pathways are associated not only with carcinogenesis, but also clinical progression of the cancer and its resistance to chemotherapy [44]. Inhibition of activated elements in these signaling pathways with the application of siRNA constitutes a potential new approach for the treatment of HPV-induced cervical cancer; each of the pathways is discussed in detail below. Figure 2. Signaling pathways altered by E6 and E7. E7
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E6 inhibits p53 that promotes apoptosis in response to DNA damage. p53 is also inactivated after binding with MDM2. E7 degrades Rb, thereby ablating Rb suppression of Akt survival pathways, resulting in activation of the antiapoptosis proteins mTor, Bcl2 and NF-κB. Akt: Protein kinase B; Bcl: B-cell leukemia; MDM: Murine double minute; mTor: Mammalian target of rapamycin; NF: Nuclear factor; Rb: Retinoblastoma.
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p53 pathway
p53 is a proapoptotic protein that is induced by stress signals such as chemotherapy and UV and other forms of radiation. It can activate both intrinsic (mitochondrial) and extrinsic apoptotic pathways. In the intrinsic pathway, p53 activates BAX, NOXA and p53-regulated apoptosis inducing protein (p53AIP)-1 [45]. In the extrinsic pathway, p53 binds to the Fas transcriptional start site and activates Fas expression [46]. E6 reduces the cellular level of p53; this phenomenon plays an important role in the carcinogenesis of cervical cancer. Inhibition of E6 increases the cellular level of p53 [47]. Thus, overexpression of p53 might be overcome by the downregulating influence of E6 and might therefore serve as a useful treatment for cervical cancer. Nutlin is a drug that dissociates p53 from mouse double minutes 2 (MDM2) protein and thus increases the cellular level of activated p53. The drug has been evaluated as a cancer treatment owing to its p53 activating properties [48]. Adenovirus-mediated wild-type p53 has been introduced to a cervical cancer cell line by transfection. In combination with radiation, it increased apoptosis [49]. Another approach is to silence MDM2 by RNAi, this was demonstrated to be effective in ovarian cancer cells [50]. Rb pathway
HPV E7 destabilizes and therefore inhibits the activity of pRb. This is evident in that inhibition of E7 by peptide nucleic acid increases activity of pRb [47]. E7 protein dissociates pRb from binding factor E2F, releasing E2F [6,51–53]. In turn, E2F activates the expression of genes encoding dihydrofolate reductases, DNA polymerases and cyclins that stimulate the cell cycle to pass from G1 to S phase [54]. E2F is overexpressed in many cancers and is linked with increased telomerase activity in immortalizing cells [55]. In mice, overexpression of E2F1 is sufficient to induce tumor formation [56]. Thus, silencing E2F might be a useful treatment for cervical cancer. In neuroblastoma, siRNA silencing of E2F has been used to inhibit euxanthone-induced neurite outgrowth [57]; however, there has been no similar experiment in cervical cancer. Protein p16, a product of the tumor suppressor gene CDKN2, plays an important role in the formation of the Rb-E2F1 complex through its effect on Rb phosphorylation [58]. When Rb is phosphorylated by G1 cyclin-dependent kinase 4 (CDK4), it dissociates from the complex and releases E2F1 [59,60]. In healthy future science group
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cells, p16 inhibits CDK4, thus reducing the release of E2F1. Deregulation of p16 is associated with cancer. In most cancers, expression of p16 is decreased, resulting in an increase in the cellular levels of CDK4 [61]. Conversely, p16 is upregulated in cervical cancer, probably due to inhibition of Rb by E7. In this instance, p16 is no longer subject to feedback inhibition from Rb [61]. Silencing of p16 expression by siRNA caused apoptosis in a cervical cancer cell line [61]. This may be related to increased levels of pRb and p53, as detected after siRNA treatment [61]. Akt
The Akt pathway, at a cellular level, is increased in cervical cancer. HPV-16 E7 can activate Akt phosphorylation through inactivation of Rb [62]. Silencing of Rb with shRNA upregulated Akt [62]. Akt promotes cellular survival through a series of target genes. For instance, Akt promotes carcinogenesis by activating mTor, NF-κB and by inactivating glycogen synthase kinase (GSK) and Bcl2antagonist of cell death (BAD). Hence, Akt activation plays an important role in the pathogenesis of HPV-induced cervical cancer, in common with many other cancers. Akt activation is also related to metastasis in cervical cancer [63]. Hence, deactivation of Akt pathways may be useful in treating HPV-induced cervical cancer. Some indication as to the potential success of this strategy can be drawn from a study in which, Akt silencing enhanced the sensitivity to chemotherapeutic drugs in a phosphatase and tensin homolog (PTEN)-mutated prostate cancer cell line [64]. mTor
A downstream protein in the Akt pathway, mTor, is upregulated in cervical cancer [65]. An mTor antagonist, rapamycin, has been used in cancer chemotherapy to enhance the efficacy of other drugs [65,66]. Oligonucleotide-mediated mTor silencing coupled to inhibition with rapamycin produces a synergistic anticancer effect in malignant glioma cells [67]. Thus, the use of siRNA to silence mTor might be effective in the treatment of cervical cancer. NF-κB NF-κB is the name for a family of inducible
dimeric transcription factors. These transcription factors are part of a larger group of DNA-binding proteins (Rel family) that recognize a common sequence motif. In cervical cancer, NF-κB is found at an abnormally high concentration in nuclei [68,69]. Such activation of future science group
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NF-κB leads to increased cellular survival, proliferation and induction of epithelial to mesenchymal transition [70]. Thus, silencing of NF-κB constitutes another potential treatment for cervical cancer. It is worth noting that NF-κB has been inhibited using siRNA in other cancers but this approach has not been studied in cervical cancer [71]. Bcl-2
Bcl-2 is an antiapoptotic protein. The Bcl-2 gene promoter contains multiple NF-κB binding sites and is thus upregulated by NF-κB. Over-expression of NF-κB in vitro leads to increased expression of the Bcl-2 protein [72,73]. In cervical cancer induced by HPV, increased expression of Bcl-2 is related to metastasis, and is ultimately, a poor prognostic in patients [63,74]. Therefore, silencing of Bcl-2 could be incorporated into future cervical cancer treatments. In chronic lymphocytic leukemia, the Bcl-2 inhibitor ABT 737 has entered clinical trials, the results of which have yet to be published. Silencing of Bcl-2 by siRNA or inhibition of Bcl-2 by ABT 737 have both been demonstrated to be effective in cancer cell lines such as hepatoma [75–77]; however, similar studies are lacking in cervical cancer. Thus, the use of siRNA to decrease the expression of Bcl-2 in cervical cancer needs to be investigated. Future of OBT for cervical cancer
We suggest that OBT that only targets E6 or E7 would not be an effective treatment, given the problems associated with the delivery of the therapy to every cancer cell and the potential for resistance. Any therapy would also need to silence signal pathways induced by E6 and E7, such as the p53 pathway (see above). Therefore any future OBT therapy would need to target several distinct molecules in a single dose. Using multiple treatment modalities on the same target might further increase the efficacy of the treatment and prevent the development of resistance. OBT might also be combined with chemotherapy and radiotherapy to maximize the treatment effect. It was previously shown that cervical cancer cells were more sensitive to chemotherapy drugs and radiation after suppression of E6 and E7 oncogenes with RNAi [10] or with a ribozyme [78]. We have shown that shRNAtreated HeLa cells with a reduced E7 protein level and reactivated p53, are more sensitive to the chemotherapy agent cisplatin [10]. Following shRNA treatment, the IC50 of cisplatin was 2.4 µM, as opposed to 9.4 µM in cells untreated 591
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with shRNA [10]. Anticancer drugs other than cisplatin now need to be investigated for their interaction with shRNA. Conclusion
The HPV vaccines, Gardasil™ and Cervarix™, have been used in many countries since 2006; 100% effective in trials, they prevent HPV infection and are estimated to reduce total cervical cancer incidence by 50%. However, to achieve this will take some 15–50 years, depending on vaccine strategies and uptake rates [79]. Clearly there is a gap in the current available treatments; even if we had full vaccine coverage today we would still require alternative treatments. Hence, we propose the development of siRNA, that target multiple signaling pathways involved in the carcinogenesis of cervical cancer, coupled to chemotherapy and radiotherapy. In combination with an effective HPV vaccination program, we might finally be able to prevent HPV-associated cervical cancer. Future perspective
We predict that a satisfactory delivery system will be developed in 1–2 years time. Within this time-frame, OBTs too will be formally
tested in clinical trials, although it is likely that some limitations will remain. For RNAi, the developed resistance and high dose requirement will then become the major issues. Should this be the case, combinational therapy with chemotherapy or radiotherapy would become more relevant. Combinational therapy with chemotherapy would be more promising especially because evermore signal pathway inhibitors will be clinically available in the near future. The pathway inhibitor closely related to the E6/p53 and E7/Rb pathways will first be considered and tested. In our view, the future treatment for cervical cancer will more likely consist of a combinational therapy rather than a single OBT treatment. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary HPV & the carcinogenesis of cervical cancer • High-risk human papillomavirus (HPV) types are responsible for 99% of cervical cancer. Continuous expression of the E6 and E7 proteins, which downregulate tumor suppressor proteins p53 and retinoblastoma (Rb) are important for carcinogenesis and cancer cell viability. Summary of oligonucleotide-based therapies for cervical cancer • Oligonucleotide-based therapies (OBTs) including antisense oligonucleotides, ribozymes and RNA interference (RNAi) can be used to specifically inhibit E6 and E7 transcription and translation and can result in inhibition of cancer cell growth and tumor growth. Limitations of oligonucleotide-based therapies in the development of treatment for cervical cancer • All OBTs face problems associated with delivery. The use of antisense oligonucleotides and ribozymes is also limited by toxicity and immunogenicity side effects. RNAi may have obstacles of developed host resistance and high dose toxicity. Inhibition of major signal pathways in the carcinogenesis of cervical cancer • Both E6 and E7 inhibit apoptosis through their downstream pathways. Except the silencing of E6 and E7, RNAi can also be used to knockout protein kinase B, mammalian target of rapamycin, B-cell leukemia-2, NF-κB and mouse double minute 2 for treatment of cervical cancer. Future of oligonucleotide-based therapies for cervical cancer • The future treatment for cervical cancer will be a combinational therapy that combines the specificity of RNAi to knockdown not only E6 and E7 but several other target genes in the E6/p53 or E7/Rb pathways with chemotherapy or radiotherapy to maximize treatment effectiveness.
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Website 101. RNAi eNewsletter Vol. 7 issue 6
www.rnai.net
Affiliations
• Aaron Irving University of Queensland, UQ Diamantina Institute, R-Wing, Princess Alexandra Hospital, Ipswich Rd, Brisbane, QLD 4102, Australia Tel.: +61 732 405 387; Fax: +61 732 405 946;
[email protected] • Nigel McMillan University of Queensland, UQ Diamantina Institute, R-Wing, Princess Alexandra Hospital, Ipswich Rd, Brisbane, QLD 4102, Australia Tel.: +61 732 405 392; Fax: +61 732 405 946;
[email protected] • Wenyi Gu University of Queensland, UQ Diamantina Institute, R-Wing, Princess Alexandra Hospital, Ipswich Rd, Brisbane, QLD 4102, Australia Tel.: +61 732 405 387; Fax: +61 732 405 946;
[email protected]
• Jiezhong Chen University of Queensland, UQ Diamantina Institute, R-Wing, Princess Alexandra Hospital, Ipswich Rd, Brisbane, QLD 4102, Australia Tel.: +61 732 405 387; Fax: +61 732 405 946;
[email protected]
www.futuremedicine.com
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